Project Summary: Sonic hedgehog (Shh) signaling is a critical developmental pathway best known to regulate cell fate specification and cell proliferation. This process requires primary cilia and involves Shh activation of downstream effector Smoothened (Smo) and Gli transcription factors (transcription-dependent). Additionally, Shh signaling regulates axon guidance in the developing nervous system as well as fibroblast migration through a distinct, transcription-independent pathway. Arl13b encodes a regulatory GTPase enriched in cilia that regulates transcription-dependent Shh signaling through Smo. Recessive mutations in ARL13B cause the ciliopathy Joubert syndrome (JS), a disorder defined by intellectual disability, cerebellar hypoplasia, and physical deformities. JS is diagnosed by MRI evidence of a hindbrain malformation known as the molar tooth sign, which is caused in part by the white matter tracts comprising the superior cerebellar peduncles (SCPs) failing to cross the midline of the brain. JS patients also exhibit midline crossing defects in the optic chiasm and the corticospinal tract. Collectively, these phenotypes indicate JS patients display axon guidance defects; however, the mechanism connecting cilia genes such as ARL13B to the regulation of axon guidance remains unclear. Because (1) Arl13b regulates transcription-dependent Shh signaling, (2) transcription-independent Shh signaling can regulate axon guidance, and (3) ARL13B mutations result in axon guidance defects in JS, we hypothesize that Arl13b regulates transcription-independent Shh signaling to direct axon guidance in the developing brain. To test this, I examined the direction and uniformity of mouse SCPs lacking either Smo or Arl13b by performing diffusion tensor imaging (DTI). I showed that SCPs lacking Smo display significant axon guidance defects in the hindbrain, indicating axon guidance in these projection neurons is Smo-dependent and suggesting Shh as a possible guidance cue. Furthermore, there is an axon guidance defect in SCPs lacking Arl13b, suggesting Arl13b regulates this transcription-independent Shh signaling pathway. Additionally, fibroblasts lacking Arl13b display decreased migration toward Shh. However, fibroblasts are able to migrate in the absence of cilia, suggesting that Arl13b may have a non-ciliary function in the regulation of transcription- independent Shh signaling. Therefore, we hypothesize that Arl13b works outside of the cilium to regulate transcription-independent Sonic hedgehog signaling to direct fibroblast migration in vitro as well as axon guidance in vivo during neural development. The aims of this project are 1) to use non-ciliary Arl13b and Smo variants to test whether Arl13b function in fibroblast migration occurs outside the cilium via modified Boyden chamber assays and 2) to define the role of Arl13b in axon guidance by examining the ability of SCP axons to cross the midline of the brain in Arl13b and Smo mouse mutants through DTI and retrograde tract tracing. Through these methods, I aim to define the role of Arl13b in transcription-independent Shh signaling and establish a novel connection between cilia-associated genes and axon guidance.